Raw qPCR Data

Data was filtered for reactions with “Call” == “Pass”. Culture condition was added to each sample to facilitate downstream analysis.

Data Points without Corresponding Culture Condition

These are samples that are not among the original sample set that were used for RNA-seq. Note the inclusion of the blank samples for the target Srp68. This indicates that the Srp68 primers amplify without template. Possible primer-dimer amplification. The Srp68 reactions should be excluded.

Summary of qPCR Data

This table contains the mean values for each culture condition and target. The key value is ddCt with is equivalent to log2 fold change. It is ddCt that will be compared to the RNA-seq estimated log2 fold changes.

Boxplot of A vs. EAN

Correlation with RNA-seq Data

AN Fit

## 
## Call:
## lm(formula = mean_ddCt ~ seq_lfc, data = corr_data %>% filter(culture == 
##     "AN"))
## 
## Residuals:
##      Min       1Q   Median       3Q      Max 
## -0.51175 -0.16423 -0.05963  0.03238  1.78971 
## 
## Coefficients:
##             Estimate Std. Error t value Pr(>|t|)    
## (Intercept)  0.05925    0.04592    1.29    0.202    
## seq_lfc      1.11003    0.05945   18.67   <2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 0.3296 on 56 degrees of freedom
## Multiple R-squared:  0.8616, Adjusted R-squared:  0.8591 
## F-statistic: 348.6 on 1 and 56 DF,  p-value: < 2.2e-16

EA Fit

## 
## Call:
## lm(formula = mean_ddCt ~ seq_lfc, data = corr_data %>% filter(culture == 
##     "AE"))
## 
## Residuals:
##     Min      1Q  Median      3Q     Max 
## -7.2250 -0.4718  0.0510  0.6835  3.4501 
## 
## Coefficients:
##             Estimate Std. Error t value Pr(>|t|)    
## (Intercept)   0.3543     0.2166   1.636    0.107    
## seq_lfc       1.2791     0.1964   6.514 2.19e-08 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 1.649 on 56 degrees of freedom
## Multiple R-squared:  0.4311, Adjusted R-squared:  0.4209 
## F-statistic: 42.43 on 1 and 56 DF,  p-value: 2.193e-08

EAN Fit

## 
## Call:
## lm(formula = mean_ddCt ~ seq_lfc, data = corr_data %>% filter(culture == 
##     "AEN"))
## 
## Residuals:
##     Min      1Q  Median      3Q     Max 
## -3.2054 -0.3782 -0.0664  0.2530  3.0005 
## 
## Coefficients:
##             Estimate Std. Error t value Pr(>|t|)    
## (Intercept)  0.34362    0.11060   3.107  0.00297 ** 
## seq_lfc      1.08462    0.04659  23.282  < 2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 0.8358 on 56 degrees of freedom
## Multiple R-squared:  0.9064, Adjusted R-squared:  0.9047 
## F-statistic:   542 on 1 and 56 DF,  p-value: < 2.2e-16

Plot of Correlation between RNA-seq and qPCR

Interactive Plot

Correlation Data

Correlation of RT-qPCR to uncorrected LFC from RNA-seq data

Correlation with uncorrected RNA-seq Data

AN Fit

## 
## Call:
## lm(formula = mean_ddCt ~ seq_lfc, data = uncor_corr_data %>% 
##     filter(culture == "AN"))
## 
## Residuals:
##      Min       1Q   Median       3Q      Max 
## -0.57507 -0.16617 -0.05160  0.04665  1.79807 
## 
## Coefficients:
##             Estimate Std. Error t value Pr(>|t|)    
## (Intercept)  0.05265    0.04669   1.128    0.264    
## seq_lfc      1.11889    0.06089  18.374   <2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 0.3342 on 56 degrees of freedom
## Multiple R-squared:  0.8577, Adjusted R-squared:  0.8552 
## F-statistic: 337.6 on 1 and 56 DF,  p-value: < 2.2e-16

EA Fit

## 
## Call:
## lm(formula = mean_ddCt ~ seq_lfc, data = uncor_corr_data %>% 
##     filter(culture == "AE"))
## 
## Residuals:
##      Min       1Q   Median       3Q      Max 
## -0.77218 -0.05822  0.02721  0.09987  0.37234 
## 
## Coefficients:
##             Estimate Std. Error t value Pr(>|t|)    
## (Intercept) -0.05990    0.02485   -2.41   0.0193 *  
## seq_lfc      1.02271    0.01166   87.68   <2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 0.1859 on 56 degrees of freedom
## Multiple R-squared:  0.9928, Adjusted R-squared:  0.9926 
## F-statistic:  7688 on 1 and 56 DF,  p-value: < 2.2e-16

EAN Fit

## 
## Call:
## lm(formula = mean_ddCt ~ seq_lfc, data = uncor_corr_data %>% 
##     filter(culture == "AEN"))
## 
## Residuals:
##      Min       1Q   Median       3Q      Max 
## -0.86008 -0.20248 -0.08165  0.05933  1.73687 
## 
## Coefficients:
##             Estimate Std. Error t value Pr(>|t|)    
## (Intercept)  0.21707    0.05683    3.82 0.000337 ***
## seq_lfc      1.00193    0.02118   47.30  < 2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 0.4268 on 56 degrees of freedom
## Multiple R-squared:  0.9756, Adjusted R-squared:  0.9751 
## F-statistic:  2238 on 1 and 56 DF,  p-value: < 2.2e-16

Plot of Correlation between RNA-seq and qPCR using uncorrected RNA-seq estimations

Environment

## R version 4.1.3 (2022-03-10)
## Platform: x86_64-apple-darwin17.0 (64-bit)
## Running under: macOS Big Sur/Monterey 10.16
## 
## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.1/Resources/lib/libRblas.0.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/4.1/Resources/lib/libRlapack.dylib
## 
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] plotly_4.10.1 ggplot2_3.4.2 stringr_1.5.0 tidyr_1.3.0   dplyr_1.1.2  
## [6] readxl_1.4.2 
## 
## loaded via a namespace (and not attached):
##  [1] tidyselect_1.2.0   xfun_0.39          bslib_0.4.2        purrr_1.0.1       
##  [5] splines_4.1.3      lattice_0.21-8     colorspace_2.1-0   vctrs_0.6.2       
##  [9] generics_0.1.3     htmltools_0.5.5    viridisLite_0.4.1  yaml_2.3.7        
## [13] mgcv_1.8-42        utf8_1.2.3         rlang_1.1.0        jquerylib_0.1.4   
## [17] pillar_1.9.0       glue_1.6.2         withr_2.5.0        RColorBrewer_1.1-3
## [21] lifecycle_1.0.3    munsell_0.5.0      gtable_0.3.3       cellranger_1.1.0  
## [25] htmlwidgets_1.6.2  evaluate_0.20      labeling_0.4.2     knitr_1.42        
## [29] fastmap_1.1.1      crosstalk_1.2.0    fansi_1.0.4        highr_0.10        
## [33] scales_1.2.1       cachem_1.0.7       jsonlite_1.8.4     farver_2.1.1      
## [37] digest_0.6.31      stringi_1.7.12     grid_4.1.3         cli_3.6.1         
## [41] tools_4.1.3        magrittr_2.0.3     sass_0.4.5         lazyeval_0.2.2    
## [45] tibble_3.2.1       pkgconfig_2.0.3    ellipsis_0.3.2     Matrix_1.5-4      
## [49] data.table_1.14.8  rmarkdown_2.21     httr_1.4.5         rstudioapi_0.14   
## [53] R6_2.5.1           nlme_3.1-162       compiler_4.1.3